鼠腦前額葉皮質對具功能性連結之中間神經元和藍斑核上而下之伴隨調控

Abstract

在這份研究中我們試圖探討中間神經元 (interneurons, IN) 是否會整合來自大腦皮質送往位在藍斑核(locus coeruleus, LC)去甲基正腎上腺素(norepinephrinergic, NE)神經細胞的資訊。在形態研究上，LC-NE神經細胞會對大腦發送全面性的投射，並作為中樞神經系統主要的NE提供者。在行為研究上，LC-NE系統能夠促進整體細胞活性(反應度、警覺度)，參與睡眠-清醒週期(sleep-wake cycle)的轉換，和與任務相關表現的提升有關。近期研究指出LC-NE系統能夠作為暫時性的過濾器，允許跟任務相關的訊息能通過感官門控(sensory gating)，進而促進行為本身，並與擁有高度認知大腦皮質所判定結果協調一致。LC神經細胞的'phasic activity'與神經網絡的增益值(gain)有高度關聯性，我們猜想phasic activity可能受到LC附近的INs調控，或是受到來自大腦皮質，如腦前額葉(prefrontal cortex, PFC)上而下的神經調節。位在PFC的腦區在評估獎賞的機制上扮演重要功能，並且這些功能與LC-NE系統的功能有高度的重疊。先前在猴子與大鼠的研究已經指出，PFC對LC的投射主要發生在核邊樹突區域(peri-cerulear dendritic zone, peri-LC)，該區域密布著LC樹突的投射。由於大部分的INs也位在peri-LC，我們猜想是否這些INs會接收PFC的投射，作為PFC和LC之間整合器。為了回答這問題，我們製作了cre酵素依賴的病毒 (AAV2-DIO-WGA)，該病毒同時擁有橫跨神經細胞突觸(trans-neuronal)的能力，藉此來揭露與LC有突觸連結的INs。我們也決定先將焦點放在研究抑制性中間神經元(inhibitory interneurons, I-IN)上面，因為在INs的群體中同時也存在著興奮性神經元(excitatory interneurons, E-IN)。AAV2-DIO-WGA被打入兩種基因型老鼠的後代，第一種老鼠為TH-cre會表現cre酵素在兒茶酚胺神經細胞(catecholaminergic neurons)中，第二種老鼠為GAD-GFP會表現綠螢光蛋白於γ-氨基丁酸神經細胞(GABAergic neurons)中，這份組合使後代能夠進行位在橋腦背側LC細胞專一性的感染，並且使目標I-INs能夠發出自體螢光。透過免疫染色，我們如預期在LC細胞上觀察到了TH和WGA的免疫活性，並在非LC細胞(沒有TH免疫活性)上觀察到了WGA的免疫活性。由於此cre酵素在兒茶酚胺神經細胞以外的細胞表現量極低，擁有很高的專一性感染可信度，我們認為這些沒有TH免疫活性、卻有WGA活性的神經細胞正是橫跨神經突觸標定到的INs，這些INs與LC細胞具有功能性連結。這些INs被發現大量聚集於LC樹突周邊的內側區域，約10.33 ± 0.12% (n=2)的INs有GFP被認定為I-INs，約22.89 ± 2.26% (n=1)的INs擁有FoxP2 轉錄因子的活性。為了證實是否這些INs接收來自大腦皮質的投射，我們重複了以上實驗並搭配了新的病毒 (AAV9-Syn-ChrimsonR-tdT)，注射於PFC以進行順行的追蹤。我們發現PFC的投射與LC和INs都有接觸。總之，這些結果支持了我們的論點，位在LC周邊樹突區域內側的E-INs和I-INs位與LC神經細胞有具功能性的連結，並且這群INs會接收、整合來自PFC送給LC神經細胞的訊息。

In this study, we aim to test whether interneurons (IN) could integrate cortical signals to norepinephrinergic (NE) neurons in locus coeruleus (LC). In morphology, LC-NE neurons sends out global axonal projections and provide major NE supply to the central nervous system. In behavior, the LC-NE system promotes general cellular activity (responsiveness, vigilance) in the brain, participating in shift of sleep-wake cycle, and relates to improvement of task-related performance. Recent studies have suggested that LC-NE system serves as a temporary filter, allowing task-related signals passing through sensory gating to facilitate behavior, in alignment of decision made by cortical areas of high cognitive function. Phasic activity of LC is highly correlated to increasing gain of neuronal network, which we suggest that it might be regulated by INs in local circuit and top-down innervating signals from cortical areas like the prefrontal cortex (PFC). Brain regions in PFC play significant roles in evaluating rewards and their functions are overlapping with those attributed to the LC-NE system. Previous studies in monkeys and rats have shown that PFC projection to LC terminates at peri-cerulear dendritic zone (peri-LC), where dendrites of LC-NE neurons are located. Since most local INs are also located in this peri-LC region, we wondered if INs received concomitant top-dwon innervations from the PFC, serving as integrators of cortical signals before passing them to LC. To answer this question, we produced a cre-dependent virus, AAV2-DIO-WGA, with trans-neuronal capacity to reveal INs synaptically connecting with LC neurons. And we decided firstly to focus on inhibitory interneurons (I-IN), besides from excitatory interneurons (E-INs). AAV2-DIO-WGA was injected into offspring of TH-cre mouse (expressing cre enzyme in catecholaminergic neurons) crossed with GAD-GFP mouse (expressing green fluorescence protein in GABAergic neurons), this allows cell-specific infection in LC neurons in dorsal pons and provides intrinsic GFP label on I-INs. Using immunohistochemistry (IHC) staining, we observed TH- and WGA- immuno-reactive (ir) within LC as expected, but also abundant neurons, which are WGA-ir but not TH-ir, in peri-LC. With high confidence of low leakage rate of cre expression in cells other than catecholaminergic population, we reason these WGA-ir and TH-negative neurons are trans-synaptically labelled and connected with LC neurons. These INs were located predominantly in the medial aspect of peri-LC region, about 10.33 ± 0.12% (n=2) of INs are found labelled with GFP being I-INs, and about 22.89 ± 2.26% (n=1) of INs with FoxP2 transcriptional factor. To confirm whether these INs receive top-down cortical inputs, we repeated experiments described above with an additional injection of AAV carrying reading frame of Crimson, AAV9-Syn-ChrimsonR-tdT, into the PFC for anterograde tracing. We found that the PFC fibers made contact with both LC neurons and INs. Together, these results support our arguments that there are local excitatory and inhibitory INs in medial peri-LC forming functional connections with LC-NA neurons and integrating the PFC inputs onto LC neurons.